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Journal of Virology, August 2001, p. 7692-7702, Vol. 75, No. 16
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.16.7692-7702.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Attenuation of Murray Valley Encephalitis Virus by Site-Directed Mutagenesis of the Hinge and Putative Receptor-Binding Regions of the Envelope Protein

Robert J. Hurrelbrink* and Peter C. McMinn

Department of Microbiology, University of Western Australia, Nedlands, Western Australia 6907, Australia

Received 12 March 2001/Accepted 16 May 2001

Molecular determinants of virulence in flaviviruses cluster in two regions on the three-dimensional structure of the envelope (E) protein; the base of domain II, believed to serve as a hinge during pH-dependent conformational change in the endosome, and the lateral face of domain III, which contains an integrin-binding motif Arg-Gly-Asp (RGD) in mosquito-borne flaviviruses and is believed to form the receptor-binding site of the protein. In an effort to better understand the nature of attenuation caused by mutations in these two regions, a full-length infectious cDNA clone of Murray Valley encephalitis virus prototype strain 1-51 (MVE-1-51) was employed to produce a panel of site-directed mutants with substitutions at amino acid positions 277 (E-277; hinge region) or 390 (E-390; RGD motif). Viruses with mutations at E-277 (Serright-arrowIle, Serright-arrowAsn, Serright-arrowVal, and Serright-arrowPro) showed various levels of in vitro and in vivo attenuation dependent on the level of hydrophobicity of the substituted amino acid. Altered hemagglutination activity observed for these viruses suggests that mutations in the hinge region may indirectly disrupt the receptor-ligand interaction, possibly by causing premature release of the virion from the endosomal membrane prior to fusion. Similarly, viruses with mutations at E-390 (Aspright-arrowAsn, Aspright-arrowGlu, and Aspright-arrowTyr) were also attenuated in vitro and in vivo; however, the absorption and penetration rates of these viruses were similar to those of wild-type virus. This, coupled with the fact that E-390 mutant viruses were only moderately inhibited by soluble heparin, suggests that RGD-dependent integrin binding is not essential for entry of MVE and that multiple and/or alternate receptors may be involved in cell entry.

* Corresponding author. Mailing address: Virology Division, TVW Telethon Institute of Child Health Research, Subiaco WA 6008, Australia. Phone: 61 8 9489 7896. Fax: 61 8 9489 7700. E-mail: robh{at}ichr.uwa.edu.au.

Journal of Virology, August 2001, p. 7692-7702, Vol. 75, No. 16
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.16.7692-7702.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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